MMV669000 (labelled PCCBTAK-0083) did not dissipate the plasma membrane Na+ gradient or increase the plasma membrane pH gradient consistent with it not inhibiting PfATP4 at the concentration tested.

The other compounds (MMV669304 (PCCBTAK-0127), MMV669360 (PCCBTAK-0156), MMV669542 (PCCBTAK-0160) and MMV669848 (PCCBTAK-0194) dissipated the plasma membrane Na+ gradient and increased the plasma membrane pH gradient at a concentration of 2 μM, consistent with them being PfATP4 inhibitors.

In the pH experiments following the addition of the test compound we then added Concanamycin A (Con A, a specific inhibitor of the parasite’s plasma membrane H+ pump) to dissipate the pH gradient. This was to verify that the phenotype was the same as that seen for the spiroindolones (see Spillman et al. Cell Host & Microbe paper).

The experiments were done by Adelaide Dennis, then a Research Assistant and now a PhD student in the Kirk lab.